Reflection microscope apparatus

ABSTRACT

An apparatus for operating an endothelium reflection microscope. The apparatus includes an optical head, which comprises: (i) an illuminating system, (ii) a frontal eye observation optical system along a central channel in which an alignment-use light spot is received and imaged by a camera having a digital optical sensor, and (iii) an enlarged-imaging optical system for enlarged observation or photographing of the subject part by the digital camera. The apparatus further comprises a motor for operating the optical head, and a CPU controller for automatically controlling the motor, the illuminating system and the frontal eye observation optical system.

This application is division of U.S. patent application Ser. No.11/632,084 filed on Jan. 8, 2007, now U.S. Pat. No. 7,726,814, theentire contents of which are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to microscopes and, moreparticularly, to non-contact endothelium microscopes and the like.

BACKGROUND OF THE INVENTION

The endothelium is the innermost layer of tissues forming the cornea,consisting of a single layer of flat polygonal cells. One purpose of theendothelium is to control water content and, thus, permit suitablehydration of the cornea. Accordingly, the shape and number of cells inthe endothelium influence the quality of one's vision. As thetransparency of the cornea depends on a rather delicate balance offactors, there are a number of diseases that can readily disrupt thisbalance, cause a loss of transparency, and, thereby, hinder the qualityof vision.

Endothelium cells in children and young people are typically hexagonalin shape. These cells, however, do not reproduce themselves. At birth,the density of endothelium cells is about 4000 per square millimeterbut, as the years pass, the cells begin to change in shape, and thetotal number of cells decreases. In an adult, the average density isabout 2700 cells per square millimeter, with a range of about 1600 toabout 3200 cells per square millimeter. The loss of endothelium cellswith age is accompanied by two main morphological changes: (i) thepresence of cells with different surface areas, and (ii) an increase inthe number of cells that are shaped differently from their originalhexagonal shape.

Evaluation of the corneal endothelium has been found useful forproviding a first clinical indication as to the potential risks ofsurgery, and for verifying a diagnosis or the effectiveness of aparticular therapy. In these evaluations, it is considered particularlyimportant to observe heterogeneous portions of the endothelium, such asintracellular and intercellular areas of no reflectance (dark spots),hyper reflective areas (bright spots), empty areas in the cells layer(guttae), bubbles, as well as Descemet's membrane rupture lines.

Such portions of the endothelium can be checked relative to theevolution of the various diseases of the endothelium which are of aninflammatory or dystrophic nature. Quantitative evaluation involves theassignment of a numeric parameter to a selected photographic field,which parameter is used to study variations in the endothelium overtime, or for comparison between different patients.

The most readily accessible parameter is the average cellular density,obtained for comparison purposes by counting the number of cellularelements. A first evaluation method, in this regard, is accomplished bycomparing the cellular dimensions with those of the hexagonal reticulesthat correspond to determined densities. According to a second method,counting of the number of cellular elements is, instead, performed byusing fixed or variable reticules.

While beneficial, neither method provides information as to theevolution of the cellular dimensions. Such information can be obtained,however, by identifying, in addition to the dimension of the averagecellular area and its variability, the perimeters of the cells as well.This information is obtained through observation using an endotheliumreflection microscope, which was first introduced in ophthalmologicpractice in 1960 by David Maurice who, by modifying a metallographymicroscope, obtained photographic images of a rabbit's cornealendothelium. Using the same principles, a microscope was developedsubsequently that was able to photograph the endothelium withoutcontacting the eye.

Generally speaking, reflection microscopes of the non-contact type arederived from high magnification microscopes with normal slit lamps.These microscopes are based on the principle of visualization of aselected structure in relation to its ability to reflect an incident rayof light used for illumination. In the most commonly used technique(i.e., triangulation), the observation angle is about 45° , themicroscope being oriented such that the bisecting axis of the angle ofview is perpendicular to the plane tangential to the corneal surface.

Non-contact endothelium microscopy is particularly suitable forapplications where contact with the cornea can be dangerous, such asimmediately after surgery or in cases where the structure of the corneais extremely fragile. By integrating the microscope with techniques ofimage analysis, the apparatus also provides a quantitative descriptionof endothelium tissue, in the form of average cellular density andspecific morphometric parameters.

In one conventional arrangement, a non-contact endothelium microscopeapparatus is provided, which includes an optical unit with anilluminating system, for obliquely illuminating through a slit a surfaceportion of a patient's eyeball, and frontal eye observation, opticalsystem, wherein an alignment-use indicator light for positionaladjustment of the imaging optical axis is projected toward the patient'seye and the resulting reflected light is received and imaged by a TVcamera. An enlarged-imaging optical system is also provided for enlargedobservation, or enlarged photographing, of the subject surface portionon the TV camera based on slit illuminating light from which the eyeballsurface has been illuminated.

In addition, a photo-detector is arranged so as to detect a position atwhich the enlarged-imaging optical system has been focused on thesubject surface portion, via a reflected optical path other than thatthrough which the enlarged image has been formed by the enlarged-imagingoptical system. The optical unit is automatically moved, in response tothe location of the indicator light displayed on a video monitor, bothin a transverse direction and toward the eye, so that the location“chases” a specified position on the screen. In this manner, when thephoto-detector detects focusing, the enlarged visual image of thesubject portion of the cornea is photographed via the TV camera.

While this system has been found workable, placement of a focusdetection, photo-detector along a supplementary reflected optical path,renders the apparatus complicated, and thus costly for providing andmaintaining reliable results.

OBJECTS AND SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide testingof the endothelium without the use of sensors, photosensors or placementof other devices in a reflected optical path.

Another object of the present invention is to provide an apparatus thatachieves a higher quality endothelium image than that of conventionalarrangements while reducing or eliminating the need for electroniccomponents and, thereby, providing greater reliability, completeness andflexibility of use.

BRIEF DESCRIPTION OF THE DRAWINGS

A specific, illustrative apparatus for morphometric analysis of thecorneal endothelium by direct image acquisition, according to thepresent invention, is described below with reference to the accompanyingdrawings, in which:

FIG. 1 shows schematically an optical pathway according to a firstembodiment of the present invention;

FIG. 2 shows schematically an optical pathway according to a secondembodiment of the present invention;

FIG. 3 illustrates schematically a hardware configuration of anapparatus according to one aspect of the present invention;

FIG. 4 shows a first image displayed on a monitor screen during imageacquisition procedures, according to one aspect of the presentinvention;

FIG. 5 shows a second image displayed on a monitor screen during imageacquisition procedures according to FIG. 4;

FIG. 6 represents schematically selected reflections obtained using anapparatus according to the present invention;

FIG. 7 is a flowchart showing a first procedure for image acquisitionusing an apparatus, according to one aspect of the present invention;

FIG. 8 is a flowchart showing a second procedure for image acquisitionusing an apparatus according to the present invention; and

FIG. 9 is a flowchart showing a third procedure for image acquisitionusing an apparatus according to the present invention.

The same numerals are used throughout the drawing figures to designatesimilar elements. Still other objects and advantages of the presentinvention will become apparent from the following description of thepreferred embodiments.

According to one aspect of the present invention, there is provided anendothelium reflection microscope apparatus having an optical headcomprising an illuminating system for obliquely illuminating, along aside projection axis through a slit, an eyeball surface of a patient'seye. The optical head also includes an eye-front observation opticalsystem along a central channel in which alignment-use indicator lightfor positional adjustment of the imaging optical center is projectedgenerally toward the eye and the resulting reflected light spot isreceived and imaged by a camera comprising a digital optical sensor. Inaddition, the optical head has an enlarged-imaging optical systemarranged along a side reflection axis for enlarged observation orphotographing of the subject part by the digital camera based on slitilluminating light with which the eyeball surface has been illuminated.The microscope further comprises a drive for moving the optical headalong three Cartesian directions including an advancement direction (Z-)generally parallel to the central channel and transverse alignmentdirections (X-, Y-), and a CPU controller for automatically controllingthe drive, the illuminating system, and the eye-front optical system.The CPU controller has a control unit operated by endothelium imageacquisition procedure software.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the drawings and, more particularly, to FIGS. 1-9,there is shown generally a specific, illustrative apparatus forexamination of the corneal endothelium and a method of operating thesame, according to various aspects of the present invention. Accordingto one embodiment, the apparatus comprises a movable optical head ormicroscope 1 having a CCD high speed camera 2, e.g., a monochromedigital camera with shooting capacity of at least one hundred frames persecond with FireWire high speed data output, such as an IEEE 1394 portor equivalent.

High speed camera 2 is connected directly to a central processing unit(CPU) 3. The CPU includes a controller 4, e.g., a 65XX type controllerproduced by National Instruments Corporation (Austin, Tex., U.S.A.) orequivalent. Controller 4 operates a power driver board 5, such that thesignal coming from the CPU is sufficient to power electric DC motors 6,as described in more detail below.

One purpose of the motors is to position microscope 1 and the associatedcamera 2, upon their automatic control by CPU 3, so that center portion7 of the eye to be examined may readily be found. This is accomplishedby reflecting light from an infrared, light emitting diode (LED) 8 intothe corneal surface, the LED being mounted to the mobile head of theapparatus, which comprises optical head or microscope 1 and camera 2.

The aforementioned electronic components are preferably connected to oneanother according to a known arrangement. Alternatively, as shown inFIG. 1, an optical scheme may be used wherein a second LED 9 withassociated optics 10 is arranged in proximity to infrared LED 8 in orderto provide a fixation point in association with a semireflecting mirror11 and a semireflecting mirror 12, as necessary, to center the patient'seye relative to the microscope and obtain the triangulation necessary toconduct the test. These components, like those forming the opticalscheme, are triangulation elements for the endothelium test, as areknown and already in use for such applications.

In accordance with one aspect of the present invention, the opticalscheme comprises a side projection axis 13, a side reflection axis 14and a central channel 15. In the embodiment of FIG. 1, a halogen lamp 16is arranged, transversely to side projection axis 13, with a lampcondenser 17 and a slit 18. Along the side projection axis, asemireflecting mirror 19 is also positioned for receiving the light beamgenerated by the halogen lamp, a beam that can be generated by a halogenlamp, and the beam of light that can be generated by a photoflash orphotoflash lamp 20 located at the beginning of side projection axis 13.On the same axis, following the photoflash lamp 20 is a photoflashcondenser 21, a slit 22 and, beyond semireflecting mirror 19, an opticalunit 23 that concentrates the beam at center portion 7 of the patient'seye. In the arrangement illustrated in FIG. 2, lamp 16, condenser 17,slit 18, semireflecting mirror 19, and photoflash lamp 20 are replacedwith a stroboscopic lamp 36 having the same function as, and activatedanalogously to, the previous optical scheme.

A side reflection optical unit 24, arranged along side reflection axis14, concentrates the reflected beam and the endothelium image on amirror 25, from which the beam and image signal are reflected to centralchannel 15 passing through a filter 26 and a magnifying optical unit 27.The beam, and the endothelium image conveyed thereby, joins the centralchannel at a point where a dichroic mirror 28 is located.

Starting from the eye to be examined, channel 15 accommodates, inaddition, semireflecting mirror 12 and a central optical unit 29 thatconcentrates the image of the eye and of LED 8 on high speed camera 2,passing through dichroic mirror 28.

The system is preferably controlled by pulses 30, 31 from controller 4.First pulse 30 transmits an on/off signal to LEDs 8 and 9, to thephotoflash lamp, and to the halogen lamp, whereas second pulse 31transmits a signal for operating motors 6.

The optical head or microscope is driven by the motors along threeCartesian directions where a low-high direction corresponds with aY-axis direction, motion in a direction horizontally approaching andmoving away from the patient's eye corresponds to a Z-axis direction,and movement in a transverse sideways direction corresponds to an X-axisdirection.

Turning now to FIGS. 4-9, the microscope, according to another aspect ofthe present invention, operates as follows. Initially, after arrangingthe optical head at a desired position, the test commences with turningon LED 9, the LED establishing a fixation point for the patient's eye.At the same time, infrared LED 8 is switched on, thereby projecting aspot of light onto the corneal surface via reflecting mirror 12. Thisspot is detected by camera 2 along central channel 15. Camera 2 thenbegins to acquire images, with a resolution of at least around 656×around 400 pixels, taken continuously at a frequency of about 100 Hz.

Desirably, data acquisition procedures are carried out with eachacquired frame to identify points (pixels) where the grey level isinside a selected predetermined range, so as to eliminate the darker andclearer points of the predetermined range, to identify all the pointsthat belong to the light spot reflected by the cornea, and thus toprecisely outline the same spot.

Of all the pixels that form the image of the reflected spot, the X and Ycoordinates are calculated, with reference to an upper left angle of theimage that coincides with the same position on the camera sensor (Seepoint ø in FIG. 4).

Subsequently, average, variance and standard deviation of the X, Ycoordinates are computed so as to define the center of the reflectedspot, and to identify the interference of possible remote luminoussignals that could be associated mistakenly with the spot.

Driver board 5 is operated continuously so that, through action ofelectronic motors 6, the luminous spot created by LED 8 follows andcoincides with the center of the camera sensor. In practice, theapparatus, according to the present invention, causes the centerposition 7 of the eye to coincide with the center of the CCD camerasensor and of the video signal processed by the FireWire IEEE 1394 portand the controller, with a feedback control loop for automatic operationof the electric motors.

More specifically, as illustrated generally in FIGS. 4 and 5, CPU 3defines two concentric areas, namely, a bigger area 32 and a smallerarea 33. The bigger area, simply stated, is the area of the image thatis deemed useful for testing purposes, the borders of the image beingdiscarded because they are often affected by undesirable externalreflections. When the center of the light spot is outside bigger area32, further testing is not permitted. Area 32 can be circular in shape,as in the example disclosed, or have a different shape (i.e., be oval,square, etc.)

The radius of area 32 may either be defined by the person operating theapparatus, or established as a design parameter, the center of the areacoinciding with the center of the CCD camera sensor. Smaller area 33, onthe other hand, is the optimal area for centering, i.e., the target areato be reached by the center of the light spot such that the eye and thecamera sensor are centered relative to one another.

In this manner, the center of the reflected spot is calculated, namely,the distance between the spot and the center of small area 33 (which caneven be as small as a single pixel). The motors are then operatedcontinuously to drive optical head or microscope 1 along the X and Ydirections until such distance is minimized, i.e., until the center ofthe reflected spot is brought (and kept) within area 33. In practice,the system automatically calculates the center location of the reflectedspot relative to the center of area 33 so as to command the motors,accordingly. Through suitable arrangement of driver board 5 and motors 6in two X-Y directions, movement of the optical head occurs at afrequency equal generally to that with which the frames are taken, i.e.,approximately every ten milliseconds.

When the reflected spot (image) is deemed centered at the sensor (Seestep A in FIGS. 7 and 8), lamp 16 is switched on through a suitable TTLsignal that activates the driver board. The lamp illuminates slit 18through lamp condenser 17, the resulting slit of light projecting on theeye along axis 13 through mirror 19 and lens 23. The optical head isthen moved along the Z-axis direction, until triangulation takes place,i.e., until the slit of light, through the geometric conditions thatregulate the optical reflection, can be reflected by the corneal surfacevia reflection axis 14. When reflection occurs, the image projected bythe slit is superimposed on the image acquired by camera 2 coming fromcentral channel 15. The aforementioned geometric conditions are suchthat advancement of the optical head in the Z-axis direction correspondsto a shifting, from left to right (See camera sensor in FIGS. 4 and 5)of the image of the slit reflected by the corneal surface.

To achieve high quality images of the endothelium, it is consideredimportant that the images be captured, and preferably that the cornea beilluminated by photoflash lamp 20, for the duration of time that theincident beam coming from side projection axis 13 is in the optimalposition to create the necessary reflection on the layer of endotheliumcells. Accordingly, the apparatus, according to one aspect of thepresent invention, operates as follows.

First, as set forth in FIG. 5, a check area or band 34 is established ina left hand side portion of the image taken by the CCD camera sensor. Inthe example shown, the check area is a band five pixels wide startingfrom the left hand border of the sensor, but may be displaced lessrelative to the center, and be smaller in width and length, depending onthe circumstances. Absent triangulation, the image in check area or band34 is generally comprised of a low intensity, grey background with a lowintensity value.

Check area 34 is checked constantly, during advancement of the opticalhead in the direction of the Z-axis, against the maximum frequencypermitted to be used with the camera (for instance, around 100 framesper second). As best seen in FIG. 6, a beam 14B is reflected by cornea Cand, more particularly, by a surface thereof, i.e., the epithelium Cep.Reflected beam 14B is captured by the camera as a luminous strip 35(i.e., the aforementioned image produced upon illumination of the slit)moving from left to right.

When luminous strip 35 enters the check area, the grey level intensitydetected increases to a value greater than a predetermined thresholdvalue; and the corresponding time t_(o) is fixed or set as a temporalreference. The grey level intensity detected in the check area isaccomplished by calculating the average intensity over all the pixelsforming the area.

From threshold or reference time t_(o), a suitable delay time Δt is setselected to control the acquisition of image data. Indeed, given thevelocity of the optical head along the Z-axis and, moreover, thethickness of the cornea, it is only with the selected delay, after image35 reflected by epithelium Cep has been detected in the check area, thatan image reflected by the endothelium arrives at an optimal position forimage capture by camera 2. An arrangement of this general description isalso shown in FIG. 6, namely, where beam 14A reflected by endotheliumCend produces a strip image 37 displaced rearwardly relative to image35, as reflected by epithelium Cep.

Generally, the length of time Δt between reference t_(o) and the timewhen the image of the endothelium is captured is fundamental, and isevaluated based on the advancement speed and the average thickness ofthe cornea. The delay time ←t can, in any case, be adjusted eithermanually or automatically. Once Δt has been reached, photoflash lamp 20is turned on, thereby illuminating the cornea, and the image of theendothelium is captured by camera 2. A number of different images can betaken, in addition, so that the one of best quality can be chosen. Theimages are then stored in a database for further processing ortreatment. After the data acquisition cycle has ended, the apparatusreturns to its starting position and awaits the next test to beperformed.

Optionally, both the time delay, Δt, and position of the check area 34can be varied so as to give to the medical operator the ability toobtain better images, particularly in the case of corneas with specificmorphologies. The photoflash lamp, with its supplementary luminousimpulse, allows the user to lower the gain of the camera for less“noise” in the images. The photoflash lamp may be actuated upon aselected advance of the optical head relative to the time lapse Δt,taking into consideration the lag intrinsic to the device.

Overall, the apparatus, according to the present invention,advantageously provides testing of the endothelium without the use ofsensors, photosensors or placement of other devices in a reflectedoptical path. It also achieves an endothelium image of much higherquality than those of conventional arrangements, while reducing oreliminating the need for electronic components, thereby providinggreater reliability, completeness and flexibility of use. The absence ofa photosensor or linear sensor along an optical reflection path, and theuse of an acquisition procedure, controlled and realized through simplesoftware-based instructions given to the apparatus, results in higherreliability, lower costs and greater flexibility. Furthermore, byallowing the user to capture a number of frames, and then choose the oneof highest quality, increases the quality of endothelium images evenmore, as compared to known arrangements and conventional focusingtechniques.

The patients, test data, and captured images are advantageously storedin a database, permitting the medical operator or user to use, reviewand/or otherwise work on the data collected, even after testing hasconcluded. In this manner, useful clinical parameters may be readilyrelied upon and, subsequently, processed so as to determine the numberand density of the cells, their shape, their surface, i.e., minimum,maximum and average surface area, their deviation from standardparameters, a variance coefficient, the ratio of cells of various forms,as well as show graphically their distribution, the dimension of cellareas, and their perimeters' distribution. Moreover, because of theautomatic control provided, testing can now be performed withsignificantly less assistance from the user.

Various modifications and alterations may be appreciated based on areview of this disclosure. These changes and additions are intended tobe within the scope and spirit of the invention as defined by thefollowing claims.

1. An endothelium reflection microscope apparatus having an optical headwhich includes an illuminating system for obliquely illuminating, alonga side projection axis through a slit, an eyeball surface of a patient'seye; an eye-front observation optical system along a central channel inwhich alignment-use indicator light for positional adjustment of theimaging optical center is projected generally toward the eye and theresulting reflected light spot is received and imaged by a cameracomprising a digital optical sensor; and an enlarged-imaging opticalsystem arranged along a side reflection axis for enlarged observation orphotographing of the subject part by the digital camera based on slitilluminating light with which the eyeball surface has been illuminated;the apparatus further comprising a drive for moving the optical headalong three Cartesian directions comprising an advancement direction(Z-) generally parallel to the central channel and transverse alignmentdirections (X-, Y-), and a CPU controller for automatically controllingthe drive, the illuminating system, and the eye-front optical system;the CPU controller including a control unit operated by endotheliumimage acquisition procedure software.